Our laboratory is studying the role of the 70-kDa heat shock proteins in various cellular processes. Specifically, we are studying the properties of the 70-kDa uncoating (UC) ATPase isolated from bovine brain which uncoats clathrin from clathrin coated vesicles. We previously found that, when the UC ATPase with bound ATP was mixed with coated vesicles, there was a stoichiometric initial burst of uncoating followed by slow steady- state uncoating. On this basis we proposed a simple model where the UC ATPase has one binding site for ATP, one for clathrin, and stoichiometric ATP hydrolysis accompanies uncoating. In contrast Schmid and Rothman proposed a more complex model with two sites for clathrin and two sites for ATP. In our current work we first confirmed that the UC ATPase has only one site for clathrin and one site for ATP. Second, we studied the ATPase activity of the UC ATPase using clathrin baskets prepared with purified clathrin and assembly proteins; clathrin baskets are a much cleaner assay system than coated vesicles which have considerable extraneous ATPase activity. Using clathrin baskets prepared with the assembly protein, AP180, we found a stoichiometric initial burst of uncoating. Associated with this initial burst of uncoating there was an initial burst of ATPase activity with about 3 ATP molecules hydrolyzed per clathrin triskelion uncoated. Thus these studies support the model of UC ATPase action which we previously proposed. Surprisingly, however, we also observed that a heat-stable protein cofactor is required for uncoating to occur. In addition to these studies on uncoating, we found that other peptide substrates of the UC ATPase bind to the same site as clathrin. Furthermore, bound nucleotide has a very specific effect on the binding of clathrin and other peptide substrates to the UC ATPase. With bound ATP, attachment and detachment of substrate occur rapidly while with bound ADP the rates of attachment and detachment are greatly decreased. Surprisingly, in the absence of bound nucleotide, the enzyme acts as if it had bound ATP rather than bound ADP suggesting that the bound ADP may alter the enzyme conformation so that substrate attachment and detachment are blocked.